Publications and other reference materials referred to herein are incorporated herein by reference and are numerically referenced in the following text and respectively grouped in the appended Bibliography which immediately precedes the claims.
The detection of small quantities of a substance may be accomplished using detectably labelled marker components. Fluorescent dyes may be used as labels in such marker components. Fluorescent dyes having greater sensitivity of fluorescent measurement are needed in order to measure substances or phenomena at lower and lower concentrations with accuracy. In fluorescence assays, the sensitivity of measurement is usually limited by the ratio of the signal obtained from a fluorescent label divided by the background signal. Accordingly, in order to provide fluorescent labels having high sensitivity one needs to minimize the magnitude of the background signal obtained and to maximize the signal from the fluorescent label.
Fluorescence assays are often used to measure substances which occur in biological materials. Such biological materials, such as serum or a cellular extract, contain a variety of components which exhibit fluorescence and may give appreciable background or ambient fluorescence which may interfere with measurement of the signal from the fluorescent label.
One way to decrease the interference from background fluorescence is to use as fluorescent labels dyes having longer emission wavelengths than the substances in a sample which give rise to background fluorescence. Most substances which constitute background fluorescence in biological materials emit in the range of about 300 to about 650 nm. For example, in human serum at a wavelength of about 725 nm background fluorescence is below the detection levels of conventionally used equipment. Fluorescent dyes having emission wavelengths which reduce interference from background fluorescence include cyanines, porphyrins and azaporphyrins. However, it has been found that the use of such labels in fluorescence assays is limited by the problems of solvent sensitivity (significantly decreased fluorescence intensity in the aqueous assay solution in comparison to dimethylformamide) and non-specific binding to biological materials.
Other properties which affect sensitivity include the magnitude of the extinction coefficient and quantum yield of the fluorescent label and its decay time. Fluorophores have a characteristic fluorescence (or "natural") lifetime, that is, the time period over which the fluorescence intensity decreases to about 37% (l/e) of its initial value in the absence of any deactivating factors. The decay time is the time period over which the decrease to the 37% (l/e) level of fluorescence is actually observed in realistic situations. Decay time may be affected by external factors, thus, a fluorophore which has a long natural lifetime may have a short observed decay time. A shortened decay time indicates deactivation of the excited state of the fluorophore and a resultingly decreased quantum yield (fluorescence quanta emitted per quantum absorbed), resulting in a smaller observed signal than is potentially there (the fluorophore loses energy as heat rather than by fluorescence emission).
Use of fluorophores having long decay times is especially important in techniques such as transient state assays where there is a need for fluorophores whose emissions may be measured over a time period of about 10 to about 20 nanoseconds. Accordingly, there is a need for fluorophores which do not suffer deactivation over such a time period.
It has been found that for fluorophores natural lifetime and extinction coefficient vary antibatically. Also, fluorophores having longer fluorescent lifetimes are more apt to be deactivated. Accordingly, fluorophores having enhanced decay times, i.e. having decay times which approach their natural lifetime, offer greater quantum yields and, thus, greater sensitivity.